Reconstitution medium for protein and peptide formulations

ABSTRACT

Compositions useful for reconstitution of concentrated formulations containing protein/peptide pharmaceuticals are provided. The composition generally includes one or more lipids, as well as one or more alcohols that promote and stabilize the formation of (a) lipid molecular assemblies with greater protein encapsulation; (b) protein-lipid complexes and (c) protein and lipid solutions. The reconstitution medium improves the protein-lipid association that in turn alters the pharmaceutical properties.

This application claims priority to U.S. provisional application No.60/492,582, filed on Aug. 5, 2003, the disclosure of which isincorporated herein by reference.

This work was funded by a grant no. RO1 HL70227-01 from the NationalInstitutes of Health. The Government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates to compositions for reconstitution offreeze-dried formulations and more particularly provides a compositionand method for reconstitution of freeze-dried formulations comprisingproteins and lipids.

BACKGROUND OF THE INVENTION

Advances in protein engineering and biotechnology, have led to largescale production of proteins and peptides for pharmaceutical purposessuch as replacement therapies and vaccines. However, due to theircomplex structure and folding dynamics, proteins undergo physical andchemical instability. These instabilities present unique difficulties inthe production, formulation, storage and administration of proteinpharmaceuticals (1,2). Chemical instability is related to covalentmodification of the protein that leads to loss of activity. One strategyto overcome this difficulty and to prolong the shelf life, is to freezedry protein products and reconstitute them prior to the administration.The reconstitution buffer is generally provided by the manufacturer.However, unique handling procedures need to be followed to avoidphysical instability, as the reconstitution involves agitation,formation of foam and froth and the exposure of proteins to air-aqueousinterface. Physical instability is related to protein folding at themolecular level, and denaturation, surface adsorption, aggregation, andprecipitation are frequent manifestations of physical instability (1-3).Such instabilities complicate the safety of protein products as thepresence of aggregates evokes undesired immune response (4). The loss ofprotein due to surface adsorption and binding to vial and syringescomplicates the therapy. In order to avoid surface adsorption, it is ageneral practice to include large quantities of albumin but inclusion ofsuch excipients presents other pharmaceutical problems including thesafety related to the source of albumin.

SUMMARY OF THE INVENTION

In order to prevent physical instability, a strategy has been proposedto add lipidic particles to stabilize the intermediate structures. Withthis invention, we report the composition of a reconstitution mediumwhich promotes the association of protein and lipid, in solution, toform stabilized lipidic molecular assemblies (cochleate, laminar, orother tertiary structures), protein-lipid bilayer complexes andprotein-lipid solutions (in which lipids associate with hydrophobicprotein domains without forming larger structures) by promotingprotein-lipid interactions. Compositions and buffer conditions forpreparing the reconstitution solution are disclosed. An example is lowconcentration of ethanol (less than about 60%, preferably 5-10% vol/vol)in various buffer systems. Another example is a solution comprising 0.5to 10 mM CaCl₂. The reconstitution of the protein in such mediumpromotes the interaction of protein with lipidic structures, improvingpharmaceutical properties such as stability,pharmacokinetic/pharmacodynamic characteristics and immune response.Such stabilized solutions have many biotechnology applications includingreplacement therapies and vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 displays the interaction of reconstitution medium with protein.The melting profile of lysozyme in the presence and absence of 20%ethanol in Phosphate buffered saline.

FIG. 2 displays the interaction of reconstitution medium with lipid. TheDifferential Scanning Calorimetry (DSC) profile of DimyristoylPhosphatidylcholine (DMPC) vesicle in the presence and in the absence of10% ethanol in phosphate buffered saline.

FIG. 3 displays the effect of reconstitution medium on both lipid andprotein. The circular dichroism (CD) melting profile of Factor VIII (5mM CaCl2 and 100 mM NaCl) in the presence and in the absence of lipid,Phosphatidylserine. The CD spectra were acquired with a heating rate of15° C./hr.

FIGS. 4 a-4 c displays the effect of reconstitution medium (5 mM CaCl2and NaCl) on lipidic structures. The LAURDAN profiles ofphosphatidylserine containing lipidic particles are shown in the absenceof calcium (4a), presence of calcium (4b) and presence of calcium andEDTA (4c).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions for media useful inthe reconstitution of dried protein formulations, and the method of useof such compositions. The compositions can be used with dry proteinpreparations, such as freeze-dried preparations containing protein toform structurally stabilized protein solutions. Furthermore, when lipidsare included, the compositions promote the interaction between theprotein hydrophobic domains and the lipid, forming solutions ofstructurally stabilized, non-aggregating, non-adhering protein/lipidsolutions.

Alcohols are frequently added as excipients in the formulation of small,conventional organic drug molecules to improve stability and solubility.Intravenous preparations containing up to 50% ethyl alcohol areavailable for human use (5-7). However, to our knowledge, alcohols arenot used in parenteral preparations which include proteins.

The present invention demonstrates that alcohols such as ethyl alcoholcan be used as excipients for protein formulations and processingcontaining lipids. It has been discovered that concentrations of alcoholwhich are below a critical concentration, roughly 60 percent by volumein the case of some common alcohols, such as ethanol, for example, havethe effect of structurally stabilizing proteins without greatly impedingtheir function. However, above the critical concentration, the alcoholinduces significant changes in secondary and tertiary structure asdemonstrated by the circular dichroism data in Examples 1 and 2,respectively. Such structural changes can be expected to affect theprotein's function. Thus, in one embodiment, the present inventionprovides a stabilized protein solution which is buffered to aphysiological pH and which comprises an alcohol at a concentration whichis less than 60% by volume and one or more proteins.

Stabilization can be determined by methods which probe secondary andtertiary structure such as the disruption of circular dichroism spectra.If circular dichroism bands due to secondary structure are undisrupted,and all of the circular dichroism spectral bands due to tertiarystructure are present, it can be inferred that little or no secondaryand tertiary structure degeneration has taken place.

Furthermore, when a lipid component is included in the formation ofsolutions which contain alcohol, the alcohol component promotes theinteraction between the protein and the lipid. Protein and lipid canassociate in numerous ways to form complexes as simple as thecombination of one or a few lipid molecules with hydrophobic proteinsites or as complex as phopholipid bilayer, cochleate and other higherorder structures. Without desiring to be bound by theory, it is thoughtthat alcohols, such as, for example, ethanol, promote the exposure ofhydrophobic domains, thereby lowering the free energy of thelipid/hydrophobic association. Moreover, protein/lipid complexes arestabilized because packing defects are induced and stabilized by alcoholmolecules (Example 4), thus decreasing the free energy of the proteinembedded in lipid complex. This effect can promote the interaction ofprotein (exposed hydrophobic domains) with lipid bilayer (packingdefects) increasing the encapsulation efficiency of proteins in lipidcomplexes. Thus, in another embodiment the present invention provides astabilized protein solution which is buffered to a physiological pH andwhich comprises one or more lipids, an alcohol at a concentration whichis less than 60% by volume, and one or more proteins.

In addition to hydrophobic force mediated protein association with lipidmolecular assemblies, the association of proteins with a specific lipidcan be a result of electrostatic-interaction This electrostaticcomponent makes some protein/lipid associations particularly stable. Forexample, FVIII and phosphatidyl serine can associate by electrostaticinteraction, and their pairing is thus particularly preferred. Thecomplex may be further stabilized by hydrophobic forces promoted by theformulations of the present invention which lead to the formation oflipid molecular assemblies such as micelles, liposomes and cochleates.

Other advantages of using alcohol as an excipient are apparent incontexts in which a protein could become denatured and aggregate insolution, posing an immunological hazard to a person to whom the proteinsolution is to be parenterally administered. Additionally, someproteins, denatured proteins in particular, have a propensity to adhereto glass and other surfaces with which they may come into contact.

It has been found that the presence of alcohol can prevent theaggregation of protein in solution and adhesion of the protein tosurfaces. The effect can be observed regardless of whether or not theprotein is denatured. For example, during the processing and storage offreeze dried protein which results in denaturation of the protein, areconstitution medium containing low alcohol concentrations may preventthe surface adsorption and aggregation of the protein. Further,denaturation during reconstitution due to agitation, shaking, formationof foam and froth and the contact of the protein with air-aqueousinterfaces may be minimized by alcohol exposure. The anti-aggregant,anti-adherent effect of the formulations of the present invention isillustrated in Example 3.

The reconstitution of the dry protein preparation may be accomplished inseveral ways. If it is desired, the dry protein preparation can becombined with a lipid component (0.3 mM to about 4 mM) by combining bothprotein and lipid, either or both in dry or solvated form before theintroduction of alcohol.

Thus in another embodiment, the present invention provides a method forreconstituting a dry protein or dry protein preparation to produce astabilized protein solution which comprises addition of lipidicparticles to said protein preparation to form a solution comprised ofprotein and lipid, and combining said solution with a reconstitutionmedium, said reconstitution medium comprising alcohol and being bufferedto a physiological pH, such that less than about 30% of the dissolvedprotein aggregates.

It may also be convenient to directly reconstitute a dry proteinpreparation which is comprised of protein and lipid which are associatedto form protein/lipid complexes, such as the product formed byfreeze-drying a solution comprised of such complexes. In a furtherembodiment, the present invention provides a method for thereconstitution of a dry protein/lipid preparation to produce astabilized protein solution comprising combining a dry proteinpreparation in which protein and lipid which are associated to formprotein/lipid complexes with a reconstitution medium, saidreconstitution medium comprising alcohol and being buffered to aphysiological pH, such that less than about 30% of the dissolved proteinforms aggregates.

In another embodiment, the reconstitution medium with and withoutethanol comprises one or more lipids and calcium ions. Suitableconcentrations of calcium are 0.5 to 10 mM and preferable concentrationis about 5 mM.

It is thought that buffer salts play a prominent role in the stabilizingof protein/lipid interactions. In particular, calcium-containing buffersplay a role in the stabilization of micellar structures, and arepreferred in cases in which micellar structures are preferred over 1)other types of complexes or 2) lower order associations. Conversely,when associative structures other than micellar structures arepreferred, such as when a solution is to be administered to a human oranimal subject and large structures would trigger an immune response, itis desirable to either lower the calcium ion concentration, or usebuffers which contain little or no calcium. The anti-aggregant,anti-adherent effects of the compositions of the present invention willbe realized with other buffers as known in the art.

By “physiological pH,” it is meant that the pH should be in the rangewhich is conducive to the functioning of biological systems, such as,for example, in the range of from about 7.0 to about 7.4. A commonphysiological pH is about 7.2.

Degree of aggregation can be determined by a number of methods. Lightscattering methods and size exclusion chromatography are examples ofmethods which can be used. Due to the range of error inherent in thesize exclusion chromatography method, the percent aggregation asdetermined by size exclusion chromatograph refers to the average valuein the range of error.

In general, the various ways of combining elements to create astabilized solution are not material to realizing the benefits of theinvention. The dried protein preparation may contain amounts of lipid,and may even be a preparation in which the lipid component is alreadyassociated with the protein as individuals/small groups or as higherorder structures such as phospholipid bilayer, lamellar or cochleatestructures. The lipid component can also be introduced either as part ofa reconstitution solution containing the alcohol component or after thedry preparation has been reconstituted with the alcohol-containingreconstitution medium. If desired, the alcohol may be added to thesolution after 1) a reconstituted protein solution has been formed, and2) the lipid component has bee added.

The dried protein preparation is most conveniently combined with theprotein as a freeze dried or lyophilized preparation. It should benoted, however, that a stabilized solution can be formed from proteinwhich has not been subjected to a drying process. Thus the benefits ofthe inventive medium extend not only to reconstituted solutions whichhave been prepared from dry protein, but also to solutions which havebeen formed from proteins introduced as emulsions, suspensions, or othernon-desiccated forms.

The proteins which can be used in the compositions and methods of thepresent invention need only have hydrophobic domains which areaccessible. By accessible, it is not meant that the hydrophobic domainsmust always be at the outer surface of the protein, but the protein musthave hydrophobic domains such that when the protein is in thereconstitution medium, the domains can be positioned, through changes inprotein conformation, such that they can contact lipids which are partof the medium. The proteins which can be used in the methods andcompositions of this invention can range greatly in size, from peptideshaving fewer than fifty amino acids and weighing several kDa, to muchlarger proteins, such as a lysozyme or FVIII, with molecular weights inexcess of 200 kilodaltons.

The lipidic molecules which can be used in the compositons and methodsof the present invention can vary widely. Phospholipids such asphosphatidyl serine and phosphatidyl choline give excellent results.However, in addition to aliphatic lipids, lipids containing groupshaving other structures, such as multi-ringed structures includingcholesterol can be used for protection against aggregation. In general,the lipid groups need only be long enough to interact with hydrophobicdomains and stabilize, through these interactions, protein conformationsin which the hydrophobic domains are exposed.

The present invention provides compositions comprising alcohols. Whenthe compositions are to be used to reconstitute formulations intendedfor administration to humans or animals, the alcohols should be suchthat they can be safely administered. Such alcohols include ethanol,glycerol and polyethylene glycol (PEG). Other alcohols, such as sugaralcohols, which can be used include sucrose, glucose, mannitol, andtrehalose. Some alcohols, such as benzyl alcohol, have been shown tohave some stabilizing effect on lipid/protein solutions, but theirtoxicity to humans makes them of questionable value, except possibly atextremely low concentrations.

The alcohol concentration is less than about 60% vol/vol, preferablyless than about 20% vol/vol, and even more preferably between about 5and 10% vol/vol, where vol/vol refers to the ratio of volume of alcoholto total solution volume (including alcohol) at standard temperature andpressure.

The protein to lipid ratio can be in the range of from about 1:10 toabout 1:1 million mol/mol. Preferred is a ratio in the range of fromabout 1:10 to about 1:100,000 mol/mol. A ratio in the range of fromabout 1:30 to about 1:10,000 mol/mol is most preferred.

Buffers can be used in concentrations in the range of from about 0.5 to600 mM, with a preferred range of from about 5 to about 600 mM for NaCland from about 0.5 to about 10 mM for CaCl₂.

In a reconstitution solution, the lipid concentration should be in therange of from about 0.3 mM to about 40 mM and preferably in the range 1mM to 10 mM.

The addition of an alcohol can have an anti-aggregant, anti-adherenteffect in solutions which are quite high in protein concentration, evenas high as about 5 mg/ml per liter. A typical example of composition isethanol (1-60% vol/vol), protein to lipid (1:30 to 1:10,000 mol/mol) andbuffer salts containing 5 mM to 600 mM NaCl and/or 0.5 mM to 10 mMCaCl₂.

The following examples of composition are given for purposes ofillustration only and not by way of limitation on the scope of theinvention. In the examples, Lysozyme was used as a model protein toinvestigate the effect of ethyl alcohol for following reasons: (i)Lysozyme is a bacteriolytic protein is under investigation as atherapeutic agent for AIDS, and (ii) detailed structural information isavailable to investigate structure-stability relationships of lysozyme.

EXAMPLE 1

We have carried out biophysical studies to determine the effect ofethanol on the secondary and tertiary structure of lysozyme as afunction of temperature. Far-UV and near-UV circular dichroism (CD)spectrophotometry was used to investigate ethanol dependent changes inconformation. Differential Scanning Calorimetry (DSC) was employed todetermine the thermodynamic parameters associated with the unfolding ofthe protein. ANS (1,8 anilinonaphthalene sulfonate), a fluorescent probethat partitions into hydrophobic domains, was used to detect theexposure of hydrophobic domains that leads to aggregation andprecipitation.

The unfolding of the protein using thermal stress in the presence and inthe absence of ethanol is carried out to investigate the thermalstability of the protein in ethanol-buffer mixtures. The lyophilizedlysozyme (660 ug/ml) was mixed with (20%) ethanol containing phosphatebuffered saline (pH 7.4) and the protein was subjected to thermalstress. The conformation and aggregation of the protein was followed byCD and turbidity measurements. As a control experiment, the lysozymedissolved in phosphate buffered saline in the absence of ethanol wasalso carried out (FIG. 1). The data shows that the presence of ethanolprevents the formation of aggregates.

In the absence of ethanol, the melting profile was distorted especiallyat temperatures around 65° C. as indicated in the figure. The turbiditymeasurements and visual inspection of the sample indicated the presenceof aggregates and precipitates of the protein. In the presence ofethanol, the analysis of unfolding profile of the protein indicated thatthe melting of the protein follows a two state model and the visualinspection of the sample suggested absence of any significant fractionof aggregates or precipitates.

In order to get further insight into the stabilization of protein in lowsolvent concentrations, we examined the conformation of the protein inthe presence of ethanol. In the absence of ethanol, the far UV CDspectrum of the protein displayed two negative bands, one around 220 nmand another more intense band around 208 nm. Qualitative analysis of thedata indicates that the secondary structural content of lysozyme ispredominantly α+β and is consistent with the 3D structure of the protein(8). At lower ethanol concentrations, (<50% v/v), a small decrease inintensity of the CD bands was observed but the shape of the spectraremained unchanged. The data clearly indicates that the lowerconcentration of ethanol did not induce substantial secondary structureof the protein. However, at higher ethanol concentrations, (>60%vol/vol), a more pronounced increase in intensity of the bands wereobserved and this intensity change was accompanied by changes in thespectral characteristics (data not shown). Overall, the ratio of thenegative bands, 220 nm to 208 nm was closer to unity in solutionscontaining higher ethanol concentration. This spectral characteristicclearly indicates that higher ethanol concentrations induced morehelical structure in the protein. The data contribute to the conclusionthat ethanol concentrations below about 60% v/v would not have anappreciably negative effect upon protein function.

EXAMPLE 2

This example describes the effect of ethyl alcohol on tertiarystructure. The near-UV CD spectrum is sensitive to the specificorientation of the aromatic groups and tertiary structure. In 100%aqueous, lysozyme displayed three positive bands at 280, 287 and 291 nm;these have been assigned to the transitions of Trp residues. In thepresence of lower concentrations of ethanol (≦60% vol/vol), enhancementin the CD bands was observed. In addition, it was also observed that theratio of the positive peaks at 280 and 287 nm was sensitive to thepresence of ethanol. However, further increase in ethanol concentrationsresulted in the loss of the CD bands, indicating a lack of anyappreciable tertiary structure (data not shown).

Based on the CD results, it is clear that the presence of ethanol atlower concentration has no effect on the secondary structure butdisplayed a slight increase in the intensity of the near UV bands. Suchincrease in near UV CD bands may possibly be due to the stabilization ofthe native state by solvents. Preferential hydration is a thermodynamicphenomenon that reflects the inability of organic molecules to interactwith the proteins and this leads to the exclusion of the organicsolvents. It has also been shown that such stabilization increases thephase transition temperature. In order to determine the mechanism ofsolvent mediated stabilization of proteins against aggregation, thermaltransition profiles were obtained (Table 1). In aqueous medium, the Tmof the protein was observed around 74.3° C. and is consistent with CDstudies and other reported results (8). Further analysis of the datarevealed that the melting temperature (Tm) and calorimetric enthalpy(ΔHcal) were dependent on ethanol concentrations; as the ethanolconcentration was increased from 0 to 20% vol/vol, both the Tm andΔH_(cal) decreased. The observed decrease in Tm may be due to thefavorable interaction of the solvent molecule with the exposedhydrophobic domains of the unfolded protein.

EXAMPLE 3

In order to determine the exposure of hydrophobic domains associatedwith the unfolding of the protein, the binding of fluorescence probessuch as 1,8 anilinonaphthalene sulfonate (ANS) was investigated. Inaqueous medium, the fluorescence intensity of the probe increased as theprotein unfolded indicating the exposure of hydrophobic domains as theprotein unfolded. The estimation of the Tm based on such profile wasaround 74° C. and is consistent with thermal denaturation studies andpreviously published results. But in the presence of low concentrationsof ethanol, the Tm and exposure of hydrophobic domains was found tooccur at lower temperature. For example, at 50° C., the fluorescenceintensity of the probe bound to protein in aqueous environment increasedby 10% over that of the unfolded state, whereas the presence of 20%ethanol the fluorescence intensity of the probe increased by 30%. Inorder to account for the contribution of solvent enhanced fluorescence,the initial fluorescence intensity of the probe was normalized and thetemperature dependent effects were calculated as percent change ratherthan absolute fluorescence intensity. The decrease in Tm and exposure ofhydrophobic domains in solvent containing solutions is possibly becauseas the protein unfolds, hydrophobic residues come into contact withclusters of solvent molecules, thus thermodynamically favoring theexposure of hydrophobic domains. But such exposure of hydrophobicdomains in aqueous environment may lead to aggregation of the protein.

Since the exposure of hydrophobic domains lead to the aggregation of theprotein, clustering of solvent molecules around the hydrophobic residuesprevent the aggregation (FIG. 1. In case the processing and storage offreeze dried protein results in denaturation of the protein, thereconstitution medium containing low solvent concentrations may preventthe surface adsorption and aggregation of the protein. Further,denaturation during reconstitution due to agitation, shaking, formationof foam and froth and the contact of the protein with air-aqueousinterfaces may be minimized by solvent exposure. The combination ofsolvent and specific ions may also promote the refolding of the proteinto the native conformation (data not shown) as the role of specific ionsin the refolding process is well documented.

The data lend support to the conclusion that the presence of smallquantities of the solvent minimize the denaturation and aggregation byclustering around the hydrophobic residues of the unfolded protein, andlow levels of alcohol can prevent aggregation of denatured protein.

EXAMPLE 4

This example describes the effect of reconstitution medium (solvent andbuffer salts) on lipid structure. In order to determine the effect ofethanol containing buffer system on the structure and dynamics ofliposomal structures, DSC thermal profiles in the presence and in theabsence of ethanol was carried out. In the absence of ethanol, the maintransition temperature was observed at 23° C. and is consistent with theprevious studies reported by others. In the presence of ethanol, thetransition peak was broadened and a shift in the temperature wasobserved. Such effects are consistent with ethanol inducing packingdefects in the bilayer organization, and thus lend support for theconclusion that ethanol induces such packing defects. The effect ofethanol on protein appears to be to expose the hydrophobic domains andit influences the packing defects in bilayer. This effect can promotethe interaction of protein (exposed hydrophobic domains) with lipidbilayer (packing defects) increasing the encapsulation efficiency ofproteins in liposomes.

EXAMPLE 5

The effect of ethanol on the bilayer structure and dynamics ofDimyristoyl Phosphatidylcholine was carried out. 6.84 mg of DMPCdissolved in chloroform, was evaporated to form a thin dry film on thewalls of a round bottom flask or Kimax tubes. The dry film wasrehydrated in 10% ethanol and phosphate buffered saline (FIG. 2). Theaddition of ethanol to DMPC vesicles resulted in the shift andbroadening of the main transition temperature indicating that ethanolcauses packing defects of the bilayer.

EXAMPLE 6

The physical stability and aggregation of 20 ug/ml of FVIII wasmonitored in the presence of PS in 25 mM TRIS, CaCl₂ and 100-300 mMNaCl. The conformation and aggregation of the protein was followed by CDmeasurements (FIG. 3). In the presence of lipid, the aggregation ofFactor VIII was reduced.

EXAMPLE 7

This example shows the effect of calcium on the formation of cochleateand bilayer structures. PS with shorter chain lipids (less that 12carbon atoms) tends to form micelles while PS with longer chain lipids(12 or more carbons) tends to form cochleates. Further, in a mixture ofPC and PS, if the proportion of PC is more, liposomes and bilayers arepreferentially formed.

This example shows fluorescence spectra acquired on an SLM 8000C or aPTI Quanta Master spectrofluorometer for cochleate and other bilayerstructures (FIG. 4). Emission spectra were acquired over the range of425 to 550 nm, using a slit width of 4 m on the excitation and emissionpaths. Correction for the inner filter effect was performed byappropriate procedures (9). Samples were maintained at the desiredtemperature using a water bath (Neslab RTE 110, NESLAB Instruments Inc,Newington, N.H.). Spectra were corrected through the use of an internalreference and further processed using software provided by themanufacturer. The effect of Ca2+ PS interaction was monitored by Laurdanfluorescence. The fluorescence emission spectra of Laurdan was sensitiveto the formation of lamellar, cochleate phases (FIG. 4). The presence ofCa2+ ions promote the formation of lipid molecular assemblies such ascochleate phases.

EXAMPLE 8

Hen egg-white lysozyme was purchased from Sigma (St Louis Mo.) as acrystallized dialyzed and lyophilized powder (Batch No: 57M7045).Recombinant FVIII was expressed in COS-7 cells and purified bychromatography. The lipids were purchased from Avanti Polar lipids(Alabaster Ala.). Spectroscopy grade solvents were purchased fromPharmaco Inc (Brookfield, Conn.) and used without further purification.ANS (1-anilino-8-naphthalene sulfonate) abd Laurdan (a probe ofhydrophobic domains, was purchased from Molecular Probes Inc. (EugeneOreg.). The ethanol-aqueous mixtures were prepared by mixing appropriatevolumes of respective solvents and the total volume estimated asdescribed in USP.

EXAMPLE 9

CD spectra were acquired on a JASCO J715 spectropolarimeter calibratedwith d10 camphor sulfonic acid. Temperature scans were acquired using aPeltier 300 RTS unit and the melting profiles were generated usingsoftware provided by the manufacturer. The spectra were acquired atheating rates of 60° C./hr and 15° C./hr. The data presented in FIG. 1is acquired using 60° C./hr heating rate and for FIG. 3, the heatingrate was 15° C./hr. For all the samples, a 10 mm cuvette was used toacquire the data. Samples were scanned in the range of 260 to 200 nm forsecondary structural analysis, and the protein concentration used was 20μg/ml (FIG. 3). For near UV CD studies, the spectra were acquired in therange of 360 to 270 nm, and the protein concentration used was 0.66mg/ml (FIG. 1). CD spectra of the protein were corrected by subtractingthe spectrum of the solvent alone, and multiple scans were acquired andaveraged to improve signal quality. The melting profiles were used todetermine the stability of the protein.

EXAMPLE 10

A two-state unfolding model was applied to analyze the equilibriumunfolding data. Each unfolding curve was normalized to the apparentfraction of the unfolded form (F_(app)), using the relationship:F _(app)=(Y _(obs) −Y _(nat))/(Y _(unf) −Y _(nat))where Y_(obs) is the ellipticity (at 220 nm or 290 nm) at a giventemperature, and Y_(unf) and Y_(nat) are the spectral values forunfolded and native structures, respectively. Y_(unf) and Y_(nat) areobtained by performing a linear regression analysis of the spectrumplateau region at high and low temperatures, respectively.

EXAMPLE 11

rFVIII clotting activity was determined by one-stage activated partialthromboplastin time (APTT) assay using micronized silica as activatorand FVIII deficient plasma as the substrate. The APTT assay wasperformed using a COAG-A-MATE coagulation analyzer (Organon TeknikaCorporation, Durham, N.C.). Briefly, rFVIII was added to FVIII deficientplasma and the clotting time was monitored. The activity of the rFVIIIwas then obtained from calibration curve constructed using the clottingtimes determined from various dilutions of a lyophilized referenceconcentrate of known activity. The concentration of the protein wasdetermined independently using Bicinchoninic acid (BCA) assay andcompared with activity. For example, all the 20-22 μg/ml of the proteincorresponds to specific activity of 87-95.6 IU. The stock solution usedto prepare the samples had a specific activity of 2174 IU/0.5 mg/ml. Thedata is shown in FIG. 3. The activity assay showed that the presence oflipidic particles did not alter the activity of Factor VIII.

EXAMPLE 12

ANS (1-anilino-8-naphthalene sulfonate) was dissolved at 1 mg/mlcontaining 2% ethanol, and a small volume was added to a solution of 10μM of lysozyme in water, to give a final probe concentration of 0.3 μM.The initial fluorescence intensity of the probe was normalized toaccount for the general solvent effects of ethanol on fluorescencemeasurements. The data indicated that the presence of ethanol results inthe exposure of hydrophobic domains of the protein.

EXAMPLE 13

Differential Scanning Calorimetric studies were carried out on aPerkin-Elmer DSC-7 instrument with samples sealed in aluminum pans. Theinstrument was calibrated with standard samples covering a wide range oftemperatures. Thermograms were recorded using a heating rate of 5°K./min (FIG. 2). For each thermogram, 14 μL of a 40 mM liposome solutionwas used. Samples were kept at the initial load temperature (15° C.) for5 minutes before each experiment. The peak area and thermodynamicparameters were calculated using software options provided by themanufacturer. Ethanol induces changes in the structure and dynamics ofbilayer structures.

EXAMPLE 14

The activity of lysozyme was determined by measuring the catalyticactivity of the protein as described earlier (10,11). The protein wasdiluted 20 times into an assay mixture containing a prefiltered cellsuspension of 0.16 mg/ml of M.lysodeikticus and the change in absorbanceat 450 nm was monitored for the bacteriolytic activity of the protein.Control experiments were performed for the relevant ethanolconcentrations and the data indicated that the presence of ethanol didnot interfere with the activity of the protein.

EXAMPLE 15

The particle size of the aggregated protein was determined using NICOMP315 particle sizer and the turbidity was measured using VARIANspectrometer. The particle size distribution was analyzed using bothGaussian and NICOMP analysis for unimodal and bimodal distribution. Thesize of the standard latex beads were measured prior to eachmeasurement. For turbidity measurements, the OD at 350 nm was followedas function of temperature.

EXAMPLE 16

The relative fraction of aggregated protein was determined using sizeexclusion chromatography (SEC). High Performance Size ExclusionChromatography (HP-SEC) was performed using Biosep SEC S4000 (4.6 mm×300mm). The analytical column was maintained at 20° C. using a ShimadzuCT0-10AC column oven. Chromatograph comprised of a Waters 510 HPLC Pump,Rheodyne injector with a 50 μl PEEK sample loop and Hitachi F1050fluorescence detector. Elution of protein was monitored using theintrinsic fluorescence of rFVIII. Excitation and emission were set at285 nm and 335 nm respectively to monitor the elution of the protein.Gel filtration was carried out under isocratic conditions at a flow rateof 0.4 ml/min using an aqueous buffer consisting of 25 mM Tris, 5 mMCaCl₂ and 300 mM NaCl, pH=7.0. The exclusion volume for the column usedeluted out at 5.1 minutes as determined using aggregated proteinstandard.

EXAMPLE 17

This example describes the formation of lipid molecular assemblies suchas micelle formation in the absence of protein after reconstitutionusing Ca2+ ions in the reconstitution medium which does not containalcohol. The formation of lipid molecular assemblies such as micelleformation were monitored using diphenyl-1-3-5-hexatriene (DPH)fluoresecnce. DPH fluorescence was measured using a PTI-Quantamasterfluorescence spectrophotometer (Photon Technology International,Lawrenceville, N.J.) at λ_(excitation) of 360 nm and λ_(emission) of 430nm. Excitation and emission slit widths were set at 2 nm. A variablepath length cuvette was used to minimize the inner filter effect.Samples were also monitored using a Submicron Particle Sizer, Nicomp-380(Particle Sizing Systems, Santa Barbara, Calif.) to determine theparticle size and detect presence of micelles at low concentrations oflipid not detectable by DPH fluorescence. Data Analysis: DPHfluorescence intensity was plotted against the concentration of lipid.Individual linear regressions were performed for the two linear portionsof the plots (before and after CMC) and linear equations were obtained.The point of intersection of these two lines is the CMC for the lipid.The CMC was obtained using the following expression:${CMC} = \frac{C_{2} - C_{1}}{m_{1} - m_{2}}$Where, C₁, C₂ are intercepts and m₁, m₂ are the slopes obtained fromeach individual regression performed on the data.

The aqueous buffer was comprised of 25 mM tris, 300 mM NaCl and 5 mMCaCl₂ (Tris buffer). Calcium chloride was excluded from the buffer forexperiments conducted with no Ca²⁺ ions. Aqueous solutions of dried DCPS(1 ml) containing various concentrations of lipid (1.0, 2.0, 2.5, 3.0,3.5, and 4.0 mM) were prepared and mixed with 1 μl of 0.005 Mdiphenyl-1-3-5-hexatriene (DPH) solution. Use of DPH as a probe tomeasure CMC depends upon its property to preferentially partition intolipidic structures (vesicles, micelles etc.) with a simultaneousincrease in the fluorescence intensity. For lipids having a tendency toform micelles, at lipid concentrations below the CMC the DPH exhibitslow fluorescence intensity which is almost independent of lipidconcentration. Beyond the CMC, DPH fluorescence intensity increases andshows a strong dependence on lipid concentration (data not shown). TheCMC of DCPS as determined using DPH in presence and absence of Ca2+ was2.30 mM and 2.96 mM respectively. Presence of calcium leads to thelowering of DCPS CMC. Also observed in presence of calcium beyond theCMC of DCPS was the formation of a white floccular precipitate whichcould be very large aggregates of DCPS micelles.

EXAMPLE 18

This example describes the effect of inclusion of protein in thecomposition of Example 17 on the formation of micelles afterreconstitution using Ca2+ ions rFVIII (10 μg/ml) was reconstituted withtris buffer containing appropriate concentrations much below CMC, 50,100 and 800 μM of DCPS. SEC profile of rFVIII shows a single broad peakat 7 minutes. Further, rFVIII can interact with DCPS (both molecular andmicellar forms) owing to its interaction with phosphoserine head groupof DCPS. Interaction of rFVIII with the molecular form of DCPS causes nosignificant changes in the molecular volume of the protein. Theinteraction of rFVIII with micellar forms of DCPS would however resultin a significant increase in the molecular volume of the protein, whichwould result in the elution of rFVIII with the exclusion volume in SEC.As determined using particle size analyzer DCPS appears to form micellesabove 1 mM concentrations. Hence, at concentrations of 50, 100 and 800μM DCPS very little change in the SEC profile of rFVIII may be expected.However the SEC profiles of rFVIII in presence of 800 and 100 μM DCPSshowed that significant amounts of rFVIII were detected in the exclusionvolume. The results from the above studies indicate that interaction ofCa2+ and rFVIII with DCPS can reduce the CMC of DCPS. This and theprevious example indicate that in the presence of protein lowerconcentrations of lipid are required to form micelles.

While this invention has been described by using specific embodiments,routine modifications to the methods and compositions described hereinwill be apparent to those skilled in the art and intended to be withinthe scope of the present invention.

REFERENCES

-   1. Ahem, T. J. & Manning, M. C. (eds.). Stability of protein    pharmaceuticals, 550 (Plenum Press, New York, 1992).-   2. Balasubramanian., S. V., Breunn, J. A. & Straubinger, R. M.    Liposomes as formulation excipient for protein pharmaceuticals; A    model protein study. Pharmaceutical Research 17, 343-349 (2000).-   3. Manning, M. C., Patel, K. & Borchardt, R. T. Stability of protein    pharmaceuticals. Pharmaceutical Research 6, 903-918 (1989).-   4. Braun, A., Kwee, L., Labow, M. A. & Alsenz, J. Protein aggregates    seem to play a key role among the parameters influencing the    antigenicity of interferon alpha (IFN-alpha) in normal and    transgenic mice. Pharm Res 14, 1472-8 (1997).-   5. Singh, M. & Ravin, L. J. Parenteral emulsions as drug carrier    systems. J Parenter Sci Technol 40, 34-41 (1986).-   6. Spiegel, A. J. & Noseworthy, M. M. Use of Nonaqueous Solvents in    Parenteral products. J. Pharm. Sci., 917-926 (1963).-   7. Wang, Y. C. & Kowal, R. R. Review of excipients and pH's for    parenteral products used in the United States. J Parenter Drug Assoc    34, 452-62 (1980).-   8. Knubovets, T., Osterhout, J. J., Connolly, P. J. &    Klibanov, A. M. Structure, thermostability, and conformational    flexibility of hen egg-white lysozyme dissolved in glycerol. Proc    Natl Acad Sci U SA 96, 1262-7 (1999).-   9. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, (Plenum    Press, New York, 1986).-   10. Rariy, R. V. & Klibanov, A. M. Protein refolding in    predominantly organic media markedly enhanced by common salts.    Biotechnol Bioeng 62, 704-10 (1999).-   11. Rariy, R. V. & Klibanov, A. M. Correct protein folding in    glycerol. Proc Natl Acad Sci USA 94, 13520-3 (1997)

1) A reconstitution medium for the reconstitution of dried proteincomprising one or more alcohols, one or more lipids, and a buffer with aphysiological pH, wherein the lipid content is in the range of fromabout 0.3 mM to about 40 mM, the buffer concentration is in the range offrom about 0.5 mM to about 600 mM and the ethanol content is less thanabout 60% vol/vol. 2) A reconstitution medium as in claim 1 wherein saidone or more alcohols has a concentration which is less than about 20%vol/vol. 3) A reconstitution medium as in claim 1 wherein said one ormore alcohols has a concentration which is in the range of from about 5%vol/vol to about 10% vol/vol. 4) A reconstitution medium as in claim 1wherein said one or more alcohols includes ethanol, polyethylene glycol,or glycerol. 5) A reconstitution medium as in claim 1 wherein said oneor more lipids includes phosphatidyl serine or phosphatidyl choline. 6)A reconstitution medium as in claim 1 wherein said reconstitution mediumincludes ethanol. 7) A reconstitution medium as in claim 5 wherein saidreconstitution medium includes ethanol. 8) A reconstitution medium as inclaim 7 containing phosphatidyl serine or phosphatidyl choline. 9) Amethod for forming a protein solution which comprises combining a mediumcomprising one or more alcohols, one or more lipids, and a buffer with aphysiological pH, wherein the lipid content is in the range of fromabout 0.3 mM to about 40 mM, the buffer concentration is in the range offrom about 0.5 mM to about 600 mM and the ethanol content is less thanabout 60% vol/vol. with a preparation comprised of a protein, whereinall of the circular dichroism spectral bands characteristic of thetertiary structure of the protein are present. 10) A method of claim 9wherein the alcohol content is less than about 20 vol/vol %, wherein allof the circular dichroism spectral bands due to tertiary structure ofthe protein are present. 11) A method of claim 9 wherein the alcoholcontent is in the range of from about 5 vol/vol % to about 10 vol/vol %,wherein all of the circular dichroism spectral bands due to tertiarystructure of the protein are present. 12) A method for thereconstitution of a protein preparation to produce a stabilized proteinsolution, said method comprising: combining a) said protein preparation,and b) a reconstitution medium, to form a reconstituted proteinsolution, said reconstitution medium comprising an alcohol and beingbuffered to a physiological pH, such that fewer than 30% of thedissolved protein forms aggregates as determined by size-exclusionchromatograph. 13) A method as in claim 12 wherein the proteinpreparation contains one or more lipids. 14) A method as in claim 12wherein said reconstitution medium contains one or more lipids. 15) Amethod as in claim 12 which further comprises adding one or more lipidsto said reconstituted protein solution. 16) A method as in claim 12wherein said stabilized protein solution contains one or more lipids.17) A method as in claim 16 wherein said alcohol is ethanol,polyethylene glycol or glycerol. 18) A method as in claim 17 wherein atleast one of said lipids is phosphatidyl serine or phosphatidyl choline.19) A method as in claim 12 wherein said protein preparation is a dryprotein preparation. 20) A method as in claim 19 wherein said dryprotein preparation contains one or more lipids which are associatedwith the protein as lipidic molecular assemblies or other protein-lipidcomplexes.